Methods and materials for treating human immunodeficiency virus infections

ABSTRACT

This document provides methods and materials for treating HIV infections. For example, methods and materials for using one or more proteosome inhibitors in combination with one or more other agents to treat HIV infections are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/552,636, filed Aug. 22, 2017 know U.S. Pat. No. 10,786,519), which isa National Stage application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2016/018776, having an International Filing Dateof Feb. 19, 2016, which claims the benefit of U.S. Provisional Ser. No.62/119,318, filed Feb. 23, 2015. This disclosure of the priorapplications are considered part of (and are incorporated by referencein) the disclosure of this application.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This inventor was made with government supposed under AI110173 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND 1. Technical Field

This document relates to methods and materials involved in treatinghuman immunodeficiency virus (HIV) infections. For example, thisdocument provides methods and materials for using one or more proteosomeinhibitors in combination with one or more other agents to treat HIVinfections.

2. Background Information

HIV is a retrovirus that causes the acquired immunodeficiency syndrome(AIDS), which is a medical condition where progressive failure of theimmune system leads to life-threatening opportunistic infections. TheHIV infection, while treatable for long periods of time, remains alargely incurable infection. On the other hand, an HIV infection was“cured” in one patient, which involved using myeloablative chemotherapyand maximally suppressive antiretroviral therapy (ART), followed by bonemarrow transplantation (BMT; Hater et al., N. Engl. J. Med., 360:692-698(2009)).

SUMMARY

This document provides methods and materials for treating HIVinfections. For example, this document provides methods and materialsfor using one or more proteosome inhibitors in combination with one ormore other agents to treat HIV infections. An obstacle to curing an HIVinfection is the existence of transcriptionally silent integrated HIVproviruses that reside in resting memory CD4 T cells, which aredifficult to eliminate because they are resistant to the cytotoxic andpro-apoptotic effects of viral reactivation. The reasons that latentlyinfected CD4 T cells resist the pro-death effects of intracellular HIVproteins are (i) memory CD4 T cells are apoptosis resistant, and (ii)chronic or latent HIV infection causes an apoptosis resistant phenotype.It is this apoptosis resistance that appears to be the reason thatlatently HIV infected cells do not die following viral reactivation,even though viral reactivation results in expression of pro-apoptoticfactors (e.g., Env, Tat, Nef, protease and Vpr, and likely othersfactors such as FasL and TRAIL).

As described herein, altering the susceptibility of latently HIVinfected cells (e.g., latently HIV infected CD4 T cells or restingmemory CD4 T cells) that have become resistant to the pro-apoptoticeffects of intracellular HIV replication, so that these cells becomesusceptible again to the pro-apoptotic effects of productive HIVreplication can be used to treat HIV infections. ART drugs do not treatlatently HIV infected cells. The only drugs that act on these latentlyHIV infected cells are latency reversing agents (LRAs), but LRAs do notcause latently HIV infected cells to die. Thus, the number of latentlyHIV infected cells can remain stable over time. As described herein,proteosome inhibitors alone such as bortezomib or ixazomib can induceHIV reactivation. Moreover, when HIV is reactivated in latently HIVinfected cells (whether the HIV reactivation is induced by a proteosomeinhibitor, another agent such as an LRA, or a condition such as aninflammatory reaction) in the presence of a proteosome inhibitor, thoseHIV reactivating cells die. Thus, in some cases, HIV infections can betreated by administering maximally suppressive ART to prevent or reducethe level of repopulation of the HIV reservoir and by administering oneor more proteosome inhibitors such as bortezomib or ixazomib to renderlatently HIV infected cells susceptible to the cytotoxic effects ofpro-apoptotic HIV proteins, to promote HIV reactivation (e.g., with aproteosome inhibitor or with an LRA), and to cause accumulation of HIVproteins within the infected cell (since proteasome inhibitors canprevent HIV budding and prevent proteasome mediated degradation of thoseproteins), altogether resulting in the intracellular expression of thepro-apoptotic HIV proteins such as Tat, Nef, Env, Vpr, and protease,which then can cause the death of those HIV reactivating cells that werelatently infected with HIV.

In general, one aspect of this document features a method for reducingthe number of latently HIV infected cells within a human infected withHIV. The method comprises, or consists essentially of, (a) administeringa proteosome inhibitor to the human, and (b) administering a combinationof anti-retroviral agents to the human. The cells can be CD4⁺ T cells.The proteosome inhibitor can be ixazomib or bortezomib. The proteosomeinhibitor can be ixazomib. The combination can comprise an integraseinhibitor, a protease inhibitor, and a reverse transcriptase inhibitor.The integrase inhibitor can be raltegravir. The protease inhibitor canbe darunavir or atazanavir. The reverse transcriptase inhibitor can beselected from the group consisting of emtricitabine, rilpivirine, andtenofovir. The method can comprise administering a latency reversingagent to the human. The latency reversing agent can be selected from thegroup consisting of an HDAC inhibitor, a phorbol ester (e.g.,prostratin), IL-2, and a bromodomain inhibitor. The method can compriseadministering an immunotherapeutic agent, a vaccine, or a nucleic acidto the human. The method can comprise administering an immunotherapeuticagent to the human, wherein the immunotherapeutic agent is IL-15. Themethod can comprise administering a vaccine to the human. The method cancomprise administering a nucleic acid to the human, wherein the nucleicacid is designed to reduce CCR5 polypeptide expression.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph plotting the percent of viable HIVIIIb infected JurkatT cells that were untreated or treated (10 or 25 nM of bortezomib) forup to five days.

FIG. 2 is a graph plotting the level of p24 (ng/mL) in the supernatantof a culture of HIV IIIb infected Jurkat T cells that were untreated ortreated (10 or 25 nM of bortezomib) as described in FIG. 1.

FIGS. 3 and 4. CD4 T cells infected with HIV-pNL4.3-Luc were activatedand cultured for three days. Cells were treated with TDF/T20 and thenwith bortezomib (10 nM) or MLN-2238 (100 nM) or control. Cells wereharvested 48 hours after treatment and analyzed for intracellularluciferase (FIG. 3) or proviral DNA (FIG. 4).

FIG. 5 is a graph plotting resting CD4 T cells treated with CCL19/IL2,infected with HIV-pNL4.3-Luc, and cultured in the presence of TDF/T20.The cells were treated with bortezomib (10 nM) or MLN-2238 (100 nM) orcontrol for 24 hours prior to reactivation with panobinostat. Cells wereharvested 48 hours after reactivation and analyzed for intracellularluciferase activity.

FIG. 6 contains flow cytometry results of J-Lat cells (a model of HIVlatency, using GFP tagged HIV) treated with nothing, bortezomib (1, 10,or 20 nM), or MLN-2238 (ixazomib; 10 or 50 nM) and analyzed 24 hourslater for activated caspase 3 expression and for HIV expression. Bothbortezomib, and ixazomib independently caused HIV reactivation asindicated by increased HIV GFP expression, and caused HIV GFP positivecells to die, as indicated by activated caspase 3 expression in the GFPpositive cells.

FIG. 7 contains flow cytometry results of CD4 T cells from an HIVpositive patient with ART suppressed HIV replication. Cells were treatedwith bortezomib or MLN-2238 (ixazomib) at the indicated doses andanalyzed 48 hours later for intracellular p24 expression (as a measureof HIV reactivation), and for activated caspase 3 expression (as ameasure of cell death). Both bortezomib, and ixazomib independentlycaused HIV reactivation as indicated by increased HIV p24 expression,and caused HIV positive cells to die as indicated by activated caspase 3expression in the p24 positive cells.

FIG. 8 contains flow cytometry results demonstrating that inhibitedbudding leads to accumulation of HIV proteins. J-Lat cells (a model ofHIV latency using GFP HIV) were treated with bortezomib or ixazomib atthe indicated doses and analyzed at 24, 48, and 72 hours. Both drugscaused HIV reactivation from latency.

FIG. 9 provides data demonstrating that proteosome inhibitors stimulateHIV. Primary CD4 T cells infected with HIV luc (top panel) or primaryCD4 T cells transfected with HIV-LTR Luc and co-transfected TK renilla(bottom panel) were treated with the indicated doses of bortezomib orixazomib, and analyzed for Luc expression as a measure of HIVreactivation. Both drugs independently reactivated HIV in primary CD4 Tcell model systems.

FIG. 10 provides data demonstrating that proteosome inhibitors stimulateATF4, leading to HIV-LTR activation. Proteosome inhibitors activated theunfolded protein response (UPR) pathway, which includes ATF-4, and ATF4can independently activate the HIV LTR. To determine if AFT4 is aplausible mechanism of HIV reactivation in primary CD4 T cells, cellswere treated with bortezomib or ixazomib, and ATF-4 was analyzed overtime.

FIG. 11. HIV latency is maintained in part by the action of HDAC, andHDAC inhibitors caused HIV reactivation. Proteosome inhibitors wereanalyzed for the ability to alter the expression of HDAC. Bortezomib andixazomib decreased HDAC-1, HDAC-3, and HDAC-4 expression in primary CD4T cells.

FIG. 12. J-LAT-10.6 or Jurkat cells were treated with bortezomib (10 nM)or ixazomib (50 nM) for the indicated times. Bortezomib and ixazomibinduced phospho PERK, phospho IRE1, and JNK activation in J Lat cells,indicating the activation of the UPR pathway.

FIG. 13. J-LAT-10.6 or Jurkat cells were treated with bortezomib (10 nM)or ixazomib (50 nM) for the indicated times. Bortezomib and ixazomibinduced ATF4 and CHOP translocation into the nucleus, and ATF6 cleavagein J Lat cells, indicating the activation of the UPR pathway.

DETAILED DESCRIPTION

This document provides methods and materials for treating HIVinfections. For example, this document provides methods and materialsfor using one or more proteosome inhibitors in combination with one ormore other agents to treat HIV infections. In some cases, one or moreproteosome inhibitors can be used to cause latently HIV infected cellsto die following HIV reactivation in those latently HIV infected cells.

Any appropriate method can be used to identify a human having an HIVinfection. For example, HIV blood tests can be used to identify a humanhaving an HIV infection.

Once identified as having an HIV infection, the human can beadministered ART (e.g., maximally suppressive ART) to prevent or reducethe level of repopulation of the HIV reservoir and one or moreproteosome inhibitors to increase the susceptibility of latently HIVinfected cells to cell death upon HIV reactivation, to induce HIVreactivation of latently HIV infected cells, and/or to causeaccumulation of HIV proteins within the reactivating cells, all of whichcan synergize to cause the death of HIV infected cells.

An ART can include any appropriate anti-retroviral agent or combinationof anti-retroviral agents. Examples of anti-retroviral agents that canbe used for ART include, without limitation, HIV integrase inhibitors,HIV protease inhibitors, and reverse transcriptase inhibitors. Examplesof ITV integrase inhibitors include, without limitation, raltegravir(also known as Isentress or MK-0518), dolutegravir, and elvitegravir.Examples of HIV protease inhibitors include, without limitation,lopinavir and atazanavir. Examples of reverse transcriptase inhibitorsinclude, without limitation, emtricitabine, rilpivirine, and tenofovir.In some cases, combinations of anti-retroviral agents can be formulatedinto a single dosage form (e.g., a single pill or capsule) such asComplera® (emtricitabine, rilpivirine, and tenofovir), Atripla®(efavirenz, emtricitabine, and tenofovir DF), Stribild® (cobicistat,elvitegravir, emtricitabine, and tenofovir), and Triumeq® (abacavir,dolutegravir, and lamivudine).

Any appropriate proteosome inhibitor or combination of proteosomeinhibitors (e.g., a combination of two, three, four, five, or moredifferent proteosome inhibitors) can be used as described herein.Examples of proteosome inhibitors that can be used as described hereininclude, without limitation, bortezomib(N-pyrazinecarbonyl-L-phenylalanine-L-leucine boronic acid; Velcade®;Millennium Pharmaceuticals), ixazomib(4-(carboxymethyl)-2-((R)-1-(2-(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-1,3,2-dioxaborinane-4-carboxylicacid, Millennium Pharmaceuticals), and carfilzomib((S)-4-Methyl-N-((S)-1-4(S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide;Kyprolis®, Onyx Pharmaceuticals, Inc.).

In some cases, one or more proteosome inhibitors can be formulated intoa pharmaceutically acceptable composition for administration to a humanhaving an HIV infection. For example, a therapeutically effective amountof bortezomib or ixazomib can be formulated together with one or morepharmaceutically acceptable carriers (additives) and/or diluents. Apharmaceutical composition can be formulated for administration in solidor liquid form including, without limitation, sterile solutions,suspensions, sustained-release formulations, tablets, capsules, pills,powders, and granules. A pharmaceutical composition containing one ormore proteosome inhibitors can be designed for oral or parenteral(including subcutaneous, intramuscular, intravenous, and intradermal)administration. When being administered orally, a pharmaceuticalcomposition containing one or more proteosome inhibitors can be in theform of a pill, tablet, or capsule. Compositions suitable for parenteraladministration include aqueous and non-aqueous sterile injectionsolutions that can contain anti-oxidants, buffers, bacteriostats, andsolutes which render the formulation isotonic with the blood of theintended recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents and thickening agents. Theformulations can be presented in unit-dose or multi-dose containers, forexample, sealed ampules and vials, and may be stored in a freeze dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules, and tablets.

In some cases, a pharmaceutically acceptable composition including oneor more proteosome inhibitors can be administered systemically. Forexample, a composition containing a proteosome inhibitor can beadministered systemically orally or by injection to a human.

Effective doses can vary depending the route of administration, the ageand general health condition of the human, excipient usage, thepossibility of co-usage with other therapeutic treatments such as use ofanti-retroviral agents and/or latency reversing agents, and the judgmentof the treating physician.

An effective amount of a composition containing one or more proteosomeinhibitors can be any amount that increases the susceptibility oflatently HIV infected cells to cell death upon HIV reactivation, incudesHIV reactivation of latently HIV infected cells, or both increases thesusceptibility of latently HIV infected cells to cell death upon HIVreactivation and induces HIV reactivation of latently HIV infectedcells, thereby causing the latently HIV infected cells to die, withoutproducing significant toxicity to the human. If a particular human failsto respond to a particular amount, then the amount of proteosomeinhibitor can be increased by, for example, two fold. After receivingthis higher amount, the human can be monitored for both responsivenessto the treatment and toxicity symptoms, and adjustments madeaccordingly. The effective amount can remain constant or can be adjustedas a sliding scale or variable dose depending on the human's response totreatment. Various factors can influence the actual effective amountused for a particular application. For example, the frequency ofadministration, duration of treatment, use of multiple treatment agents,route of administration, and severity of the HIV infection may requirean increase or decrease in the actual effective amount administered.

The frequency of administration of a composition containing one or moreproteosome inhibitors can be any frequency that increases thesusceptibility of latently HIV infected cells to cell death upon HIVreactivation, incudes HIV reactivation of latently HIV infected cells,or both increases the susceptibility of latently HIV infected cells tocell death upon HIV reactivation and induces HIV reactivation oflatently HIV infected cells, thereby causing the latently HIV infectedcells to die, without producing significant toxicity to the human. Forexample, the frequency of administration can be from about daily toabout once a week. The frequency of administration can remain constantor can be variable during the duration of treatment. As with theeffective amount, various factors can influence the actual frequency ofadministration used for a particular application. For example, theeffective amount, duration of treatment, use of multiple treatmentagents, route of administration, and severity of the HIV infection mayrequire an increase or decrease in administration frequency.

An effective duration for administering a composition containing one ormore proteosome inhibitors can be any duration that increases thesusceptibility of latently HIV infected cells to cell death upon HIVreactivation, incudes HIV reactivation of latently HIV infected cells,or both increases the susceptibility of latently HIV infected cells tocell death upon HIV reactivation and induces HIV reactivation oflatently HIV infected cells, thereby causing the latently HIV infectedcells to die, without producing significant toxicity to the human. Thus,the effective duration can vary from several months to several years. Ingeneral, the effective duration for the treatment of an HIV infection asdescribed herein can range in duration from about two months to aboutfive years. Multiple factors can influence the actual effective durationused for a particular treatment. For example, an effective duration canvary with the frequency of administration, effective amount, use ofmultiple treatment agents, route of administration, and severity of theHIV infection being treated.

In some cases, a human having an HIV infection can be treated with oneor more proteosome inhibitors as described herein in combination with(a) one or more anti-retroviral agents, (b) one or more latencyreversing agents, (c) one or more immunotherapeutic agents, (d) one ormore vaccines (e.g., a vaccine formulated for assisting in the treatmentof an HIV infection), (e) one or more nucleic acid-based therapies, (f)one or more chimeric TRIMS a polypeptides designed to restrict HIVexpression, and (g) one or more advanced (e.g., third or latergeneration) chimeric antigen receptors expressed on CD8 T cells or NKcells designed to generate anti-HIV immunity. Examples of latencyreversing agents that can be used in combination with one or moreproteosome inhibitors as described herein include, without limitation,HDAC inhibitors, phorbol esters, IL-2, bromodomain inhibitors, and thosedescribed elsewhere (Bullen et al., Nature Medicine, 20:425-429 (2014)).Examples of HDAC inhibitors that can be used as latency reversing agentsinclude, without limitation, vorinostat, panabinostat, and valproicacid. Examples of phorbol esters that can be used as latency reversingagents include, without limitation, prostratin and PMA. An example of abromodomain inhibitor that can be used as a latency reversing agentincludes, without limitation, JQ1 ((S)-tert-butyl2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate).

Examples of immunotherapeutic agents that can be used in combinationwith one or more proteosome inhibitors as described herein include,without limitation, IL-15, CD4 immunotoxin, and neutralizing anti-HIVantibodies. For example, a human having an HIV infection can beadministered one or more proteosome inhibitors as described herein andIL-15.

Examples of vaccines that can be used in combination with one or moreproteosome inhibitors as described herein include, without limitation,HIV tat or env antigens delivered by any number of platforms includinggenetic immunization, viral or virus like particle delivery, or deliveryas recombinant proteins. The HIV antigens can be delivered withadjuvants such as CPG or GM-CSF. In some cases, a human having an HIVinfection can be administered one or more proteosome inhibitors asdescribed herein and a HIV tat or env vaccine.

Examples of nucleic acid-based therapies that can be used in combinationwith one or more proteosome inhibitors as described herein include,without limitation, nucleic acid molecules having the ability to reduceCCR5 polypeptide expression (e.g., siRNA molecules designed to reduceCCR5 polypeptide expression) and TALEN or CRISPR/Cas constructs designedto excise HIV DNA. For example, a human having an HIV infection can beadministered one or more proteosome inhibitors as described herein andan siRNA molecule designed to reduce CCr5 polypeptide expression.

In some cases, a human having an HIV infection can be treated with oneor more proteosome inhibitors as described herein in combination withone or more anti-retroviral agents plus any one or more of (a) one ormore latency reversing agents, (b) one or more immunotherapeutic agents,(c) one or more vaccines (e.g., a vaccine formulated for assisting inthe treatment of an HIV infection), and (d) one or more nucleicacid-based therapies.

In some cases, the level of HIV infected cells within a human beingtreated can be monitored during the course of treatment. Any appropriatemethod can be used to determine the level of HIV infected cells within ahuman. For example, the level of HIV infected cells within a human canbe assessed using PCR based detection methods (nested or un-nested) fordetecting HIV DNA, quantitative viral outgrowth assays (QVOA) formeasuring replication competent HIV levels, or TILDA (Tat/rev; InducedLimiting Dilution Assay) that can measure the frequency of cells withmultiply spliced HIV RNA as a surrogate for replication competent HIV.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Using Proteosome Inhibitors to Cause Latently HIVInfected Cells to Die Following HIV Reactivation

HIV infection is effectively treated by a number of antiretroviralmedication, which when used in well proven combinations effectivelysuppress HIV replication, and persons on these treatments no longerexperience life threatening immunosuppression associated with HIVinfection. Even though combination antiretroviral therapy reverses theimmunodeficiency of HIV, it does not normalize health of infectedindividuals. For example, treated HIV patients experience acceleratedrates of diseases associated with ageing, likely as a consequence ofon-going inflammation, including stroke, heart disease, diabetes, andosteoporosis (Warriner et al., Infect. Dis. Clin. North Am.,28(3):457-76 (2014)).

A principal obstacle to curing an HIV infection is the existence of along lived population of resting memory CD4 T cells, in which HIV hasintegrated into the host genome, where it lays dormant, and thus doesnot express any HIV proteins. Thus, current antiretroviral agents, whichtarget HIV encoded proteins do not target these cells, nor do anti-HIVspecific immune response as no target antigen is expressed. Current ARTdoes, however, efficiently control and reduce HIV replication (reflectedby HIV plasma RNA levels) to undetectable levels, such that in awell-treated patient, the majority of HIV persists in the latent(integrated DNA) state. The quiescent nature of this reservoir isassociated with a long half-life of latently HIV infected CD4 T cells(estimated at about 44 months), such that a predicted 70 years oftherapy would be required to cure HIV assuming that no HIV reactivationwould occur during that time frame. Of course, however HIV reactivateswith concomitant inflammation for example during “colds” or withvaccinations, and these viral “blips” serve to repopulate the HIVreservoir. Thus, the size of the HIV reservoir can be remarkable stableover years, and to date there have been no interventions identifiedwhich reliably reduce the size of the HIV reservoir. Importantly, whenHIV reactivates from latency, those reactivating cells do not die. Asdescribed herein, when latently HIV infected cells are treated withproteasome inhibitors, those cells which reactivate HIV die, whereascells which do not contain HIV are spared.

Latently infected CD4 T cells are rare in HIV infected and treatedindividuals, being present at a frequency of about 1 per million CD4 Tcells. Moreover, as HIV is latent, there are no cell surface markersthat can be used to identify latently infected cells. Thus, isolation,purification, and study of these cells using ex vivo samples ispractically impossible. Accordingly, the HIV field developed a number ofin vitro models to recapitulate many of the properties of latency. Onemodel of HIV latency uses CCL19, which is the ligand of CCR7, topolarize cells towards a resting memory and thus a latency resemblingphenotype. Pretreatment of resting CD4⁺ T cells from blood with CCL19allows for efficient HIV-1 entry and viral integration with restrictedspontaneous viral expression yet robust HIV expression post stimulationconsistent with postintegration HIV-1 latency (Saleh et al., Blood,110:4161-4164 (2007)).

ART drugs do not treat latently HIV infected cells. The only drugs thatact on these latently HIV infected cells are latency reversing agents(LRAs), but LRAs do not cause latently HIV infected cells to die. Thus,the number of latently HIV infected cells can remain stable over time.

The results provided herein (see, e.g., FIGS. 1-13) demonstrate thatproteosome inhibitors alone such as bortezomib or ixazomib can induceHIV reactivation and that, following HIV reactivation, HIV proteinsaccumulate in the HIV infected cell. Moreover, the results providedherein demonstrate that when HIV is reactivated in latently HIV infectedcells (whether the HIV reactivation is induced by a proteosomeinhibitor, another agent such as an LRA, or a condition such as aninflammatory reaction) in the presence of a proteosome inhibitor, thoseHIV reactivating cells die, resulting in fewer cells containing HIV.

Example 2 Reducing the Number of Latently HIV Infected Cells within anHIV Infected Human

A human having an HIV infection is treated with an ART regimen thatincludes 400 mg po bid of Raltegravir or 50 mg per day of Dolutegravirplus either two nucleoside analogues such as tenofovir (300 mg per day)and emtracitibine (200 mg per day or Complera® (emtricitabine,rilpivirine, and tenofovir; fixed dose pill once daily). In addition,the human is treated with 2 mg/m² of ixazomib twice a week.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for increasing apoptosis susceptibilityof latently HIV infected cells within a human infected with HIV, whereinsaid method comprises administering ixazomib to said human to contactlatently HIV infected cells with 50 nM to 100 nM of said ixazomib,thereby increasing the susceptibility of latently HIV infected cellswithin said human to pro-apoptotic HIV proteins.
 2. The method of claim1, wherein said infected cells are resting memory CD4⁺ T cells.
 3. Themethod of claim 1, wherein said method further comprises administering alatency reversing agent to said human to treat HIV.
 4. The method ofclaim 3, wherein said latency reversing agent is selected from the groupconsisting of an HDAC inhibitor, a phorbol ester, IL-2, and abromodomain inhibitor.
 5. The method of claim 1, wherein said methodfurther comprises administering an immunotherapeutic agent, a vaccine,or a nucleic acid to said human to treat HIV.
 6. The method of claim 1,wherein said method further comprises administering an immunotherapeuticagent to said human to treat HIV, wherein said immunotherapeutic agentis IL-15.
 7. The method of claim 1, wherein said method furthercomprises administering a vaccine to said human to treat HIV.
 8. Themethod of claim 1, wherein said method further comprises administering anucleic acid to said human to treat HIV, wherein said nucleic acid isdesigned to reduce CCR5 polypeptide expression.